The function and half-life of most cytoplasmic and nuclear proteins, as well as some membrane proteins, is regulated by the covalent linkage of ubiquitin. Ubiquitination a key regulator of degradation by the proteasome. In neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease, ubiquitinated proteins accumulate, indicating a possible dysregulation of ubiquitination or proteasome system. Mutations in enzymes involved in ubiquitination have been associated with familial forms of neurodegenerative disease, including loss of function mutations in parkin for Parkinson's disease. In this application, we propose a proteomic approach to study ubiquitination under normal and disease-like toxic settings. As a first step, we have generated a mouse L cell line that stably expresses a hexa-histidine and GFP tagged ubiquitin protein and demonstrated that this modified ubiquitin can be conjugated to protein targets and then purified on immobilized metal affinity chromatogrphy (IMAC). Proteins purified in this manner can be directly anlayzed by MALDI-TOF and tandem mass spectroscopy to obtain sequence information. We now propose 3 Aims that are designed to characterize protein substrates of ubiquitination and to study how certain types of insults affect the process. In Aim 1, we will study the mouse L cells to characterize the following; 1) steady-state ubiquitinated protein profiles, 2) cell cycle specific profiles, 3) profiles after oxidative insult, 4) profiles after serum deprivation, 5) profiles after heat shock, and 6) profiles after induction of the unfolded protein response. In Aim 2, we will construct lines of human neuroblastoma SY5Y and astrocytoma CCF-STTG1 cells that stably express hexa-his-GFP-Ubq for a similar characterization. Collectively, these studies will lead to a better characterization of the substrates of ubiquitination and how various disease-like insults alter the process. We anticipate that some of the newly identified proteins may be useful biomarkers for certain types of toxicity in vivo.